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V. S. Yellore, I. N. Lai, S. A. Rayner, A. J. Aldave; Demonstration of the Utility of Next Generation DNA Resequencing in the Identification of the Genetic Basis of Posterior Polymorphous Corneal Dystrophy. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1645.
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© ARVO (1962-2015); The Authors (2016-present)
Posterior polymorphous corneal dystrophy 1(PPCD1) has been mapped to overlapping regions of chromosome 20 in four families, although Sanger sequencing of the coding regions of the 26 positional candidate genes mapped to the common support interval did not reveal a presumed pathogenic mutation. As we believe that the causal variant is most likely a coding region deletion or a variant in a noncoding region of the PPCD1 common support interval, we performed next generation resequencing of the common PPCD1 interval in members of a family with PPCD1.
Enrichment of the portion of chromosome 20 containing the common PPCD1 interval and Next Generation Resequencing using the Roche 454 Titanium platform was performed on DNA extracted from an affected and an unaffected member of a family previously linked to the PPCD1 locus. Computational analysis using NextGene Software was performed, including screening for exonic sequence variants previously identified with Sanger sequencing.
Next generation resequencing of the selectively enriched chromosomal 20 region between markers D20S48 and D20S471 produced over 290,000 DNA sequence reads with an average of 370 bases for each of the two DNA samples. Alignment of the DNA sequence reads with the reference sequence from the public database resulted in over 66 million matched bases per sample. Analysis of the matched bases demonstrated 5X coverage of the enriched chromosome 20 sequence with approximately 37,000 DNA sequence variants in each sample that mapped to chromosome 20. Of these sequence variants only 1% represented coding region variants. 24 of these sequence variants lay within the exons of 11 genes that map to the common PPCD1 interval and were identified by the next generation resequencing analysis.
Next generation resequencing of DNA from a PPCD patient and his unaffected son generated a large amount of sequence information. Mining of this data revealed all 24 sequence variants identified previously by Sanger sequencing within exonic regions. However the data contains a very large number of sequence variants within non-coding regions that need to be further analyzed for their possible role in PPCD1.
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